Methods for synthesizing cDNAs are essential techniques for researches in the fields of medical science and biology as an indispensable method for analyzing gene transcripts. Any DNA genetic information manifests physiological activity through transcripts and a potential means for analyzing such transcripts is cDNA cloning. In cDNA syntheses according to conventional methods, clones are ultimately isolated from a cDNA library synthesized from poly A sites by using oligo dT as a primer. However, in most cases using such a method, whole structures of transcription units cannot be analyzed since the transcription units are not synthesized in their full-lengths. Therefore, when using a conventional cDNA library, it is essential for analysis of gene structures in their full-lengths to synthesize 5' upstream regions by the primer elongation method, or perform gene-walking of the 5' upstream regions by cDNA synthesis using a random primer.
However, such conventional methods for synthesizing cDNAs as described above have, for example, the following problems.
1. cDNAs covering most part of transcripts can be obtained by using a random primer. However, those cDNAs are short fragments and clones covering from the poly A site to 5' Cap site cannot be isolated. PA0 2. Any cDNAs obtained by using oligo dT as a primer contain the 3' end. However, because the reverse transcriptase cannot reach the 5' Cap site, the 5' upstream should be further isolated and analyzed by the primer elongation method and 5' RACE or the like. PA0 3. Efficiency of any conventional methods for isolating cDNAs in their full-lengths including those methods mentioned above is not sufficient (only 2,000,000 recombinant phages can be obtained from 100 .mu.g of mRNA). Therefore, more efficient techniques are desired for practical purposes. PA0 1 Because the recognition of 5' Cap site depends on reactions of proteins like adenovirus Cap binding protein and the de-capping enzyme of tobacco mosaic virus, high efficiency of the selection of full-length cDNAs (RNAs) cannot be expected. PA0 2 When the first strand of cDNA is synthesized by a reverse transcriptase, the synthesized strand does not extend to the 5' Cap site. PA0 3 There are also problems of the addition of primer sequences, synthesis efficiency of second strand, cloning efficiency of double stranded cDNA after the synthesis of the first strand, and of a host vector system for cloning.
As conventional methods for synthesizing full-length cDNAs, the following methods can be mentioned;
the method utilizing a Cap binding protein of yeast or Hela cells for labeling the 5' Cap site (I. Edery et al., "An Efficient Strategy To Isolate Full-length cDNAs Based on a mRNA Cap Retention Procedure (CAPture)", MCB, 15, 3363-3371, 1995); the method where phosphates of incomplete cDNAs without 5' Cap are removed by using alkaline phosphatase and then the whole cDNAs are treated with de-capping enzyme of tobacco mosaic virus so that only the full-length cDNAs have phosphates (K. Maruyama et al., "Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides", Gene, 138, 171-174, 1995., S. Kato et al., "Construction of a human full-length cDNA bank", Gene, 150, 243-250, 1995) and the like.
The reasons why efficiency of these conventional methods for synthesizing full-length cDNAs is not sufficient include, for example, the followings.
As described above, in the production of cDNA libraries in a multi-step process, there are problems such as those mentioned as 1 to 3 above.
Therefore, the first object of the present invention is to provide a novel method in which 5' Cap site can be more efficiently labeled compared with the labeling by the proteins reactions such as those by the conventional adenovirus Cap binding protein and the de-capping enzyme of tobacco mosaic virus which is directed to isolation of full-length cDNAs.
The second object of the present invention is to provide a method for making full-length cDNA libraries utilizing the novel method for labeling of the 5' Cap site. The inventors of the present invention have found a novel method for preparing a full-length cDNA libraries, and have applied for patent ahead (Japanese Patent application No. Hei 8-60459/1996).
By this method the labeling of the 5' Cap site is more efficiently performed compared to the protein enzyme reaction with the above adenovirus Cap binding protein such as Cap binding protein and Tabbaco mosaic virus, and as a result, the preparation of the full-length cDNA libraries become more easy.
However, as the inventors further studied on this method, it has been found that mRNA tends to be cleaved during a procedure of dialdehydration of a diol structure, and an efficiency of a synthesis of a full-length cDNA is decreased.
Therefore, the object of the present invention is to prepare full-length cDNA libraries utilizing a method for labeling the 5' Cap site more efficiently than protein enzyme reactions, which avoids a decrease of a full-length cDNA synthesis efficiency caused by cleavage of mRNA, and can synthesize a full-length cDNA more efficiently.